Breast cancer is one of the major cancers occurring in woman. About 60% of premenopausal and 75% of postmenopausal patients have estrogen-dependent carcinomas as identified by the presence of estrogen receptor in tumor biopsies. Deprivation estrogens in such patients results in tumor regression. Aromatase is the rate-limiting enzyme in estrogen biosynthesis. In addition, aromatase activity has been measured in the estrogen-dependent breast tumor. Consequently, it is considered that this enzyme may play a key role in the pathogenesis of estrogen-dependent breast cancer and in its treatment. Aromatase inhibitors, such as aminoglutethiomide and 4-hydroxyandrostene-3, 17-dione, have been used in clinical trials and investigations. The long term objectives of this project are two folds. First, we plan to obtain structural information concerning aromatase, especially around the active site region. The structural information will be important for designing ideal aromatase inhibitor for treatment of breast cancer. Second, we will apply molecular biology techniques to study the genetic and molecular mechanism responsible for this existence of aromatase in breast cancer. An human aromatase cDNA has been recently characterized in this laboratory. A full-length aromatase cDNA will be constructed using our current cDNA clone. Furthermore, a method for an expression of the full length aromatase cDNA on nonsteroidogenic cells COS 1 cells, will be developed. This expression study will set the stage for our next phase of research, to apply site-directed mutagenesis in the investigation of the structural features involved in the enzyme's catalytic properties. Though Southern blotting experiments, two aromatase genes in human genome have been indicated. These two genes for aromatase will be characterized by studying two aromatase genomic clones recently isolated in this laboratory. In addition, polyclonal antibodies against aromatase have been prepared. These DNA probes and antibodies will be used to determine whether the induction of aromatase in breast tumors takes place at the transcriptional or translational level, or by enhancing the specific activity of the enzyme.